Pre-mRNA processing enhancer and method for intron-independent gene expression

ABSTRACT

A chimeric RNA molecule comprising at least one pre-mRNA processing element is disclosed. A gene construct comprising a DNA sequence encoding at least one pre-mRNA processing enhancer is also disclosed. A method of enhancing cytoplasmic RNA accumulation is disclosed. This method comprises the step of inserting a DNA sequence encoding the RNA into the vector described and expressing the DNA sequence.

FIELD OF THE INVENTION

In general, the field of the present invention is methods ofintron-independent gene expression. Specifically the field of thepresent invention is use of pre-mRNA processing enhancers to enhancecytoplasmic accumulation of intronless RNA molecules.

BACKGROUND

Formation of mature mRNAs in higher eukaryotes requires that severalprocessing steps occur in the nucleus prior to transport of the mRNA tothe cytoplasm. For intron-containing transcripts, these steps include5'-cap formation, methylation, 3'-end cleavage and polyadenylation, andsplicing. Intronless transcripts do not undergo splicing but, in mostcases, still need to undergo these other steps in pre-mRNA processing.Much information has been obtained concerning the biochemistry andmachinery involved in these nuclear processing events (see Green, Annu.Rev. Cell. Biol. 7:559-599, 1991, for review). Nevertheless, therelationship of these events to nuclear export and cytoplasmicaccumulation remains poorly understood.

The first evidence linking splicing and the accumulation of mRNA in thecytoplasm came from studies with SV40 (Gruss, et al., Proc. Natl. Acad.Sci. USA 76:4317-4321, 1979). Cells transfected with SV40 mutantslacking an excisable intron in the late region of the viral genome werefound to synthesize late transcripts, but not to accumulate late SV40mRNA in the cytoplasm. The requirement of an intron for efficientcytoplasmic accumulation of mRNA (i.e., intron-dependent geneexpression) has been subsequently demonstrated for many other genes aswell, including those encoding β-globin (Hamer and Leder, Cell17:737-747, 1979; Buchman and Berg, Mol. Cell. Biol. 8:4395-4405, 1988;Ryu, Processing of transcripts made from intron-containing andintronless protein-coding genes, Ph.D. Thesis, University ofWisconsin-Madison, Madison, Wis., 1989; Collis, et al., EMBO. J.9:233-240, 1990), ribosomal protein L32 (Chung and Perry, Mol. Cell.Biol. 9:2075-2082, 1989), purine nucleoside phosphorylase (PNP)(Jonsson, et al., Nucleic Acids Res. 20:3191-3198, 1992), immunoglobulinμ (Neuberger and Williams, Nucleic Acids Res. 16:6713-6724, 1988), mousethymidylate synthase (Deng, et al., Mol. Cell. Biol. 9:4079-4082, 1989),mouse DHFR (Gasser, et al., Proc. Natl. Acad. Sci. USA 79:6522-6526,1982), plant alcohol dehydrogenase-1 (Callis, et al., Genes & Dev.1:1183-1200, 1987), and triosephosphate isomerase (TPI) (Nesic, et al.,Mol. Cell. Biol. 13:3359-3369, 1993). It has been proposed that thepresence of introns can protect pre-mRNAs from degradation in thenucleus (Hamer and Leder, 1979, supra; Buchman and Berg, 1988, supra;Ryu and Mertz, J. Virol. 63:4386-4394, 1989), facilitate polyadenylation(Collis, et. al., 1990, supra; Huang and Gorman, Nucleic Acids Res.18:937-947, 1990; Niwa, et al., Genes & Dev. 4:1552-1559, 1990; Pandey,et al., Nucleic Acids Res. 18:3161-3170, 1990; Nesic, et al., 1993,supra; Ryu, et al., manuscript in preparation, 1995), facilitateexcision of an adjacent intron (Ryu, 1989, supra; Nesic and Maquat,Genes & Dev. 8:363-375, 1994), and target mRNAs for export to thecytoplasm (Hamer and Leder, 1979, supra; Buchman and Berg, 1988, supra;Chang and Sharp, Cell 59:789-795, 1989; Legrain and Rosbash, Cell57:573-583, 1989; Ryu and Mertz, 1989, supra).

Because of this intron requirement, the complementary DNA (cDNA) versionof most genes is expressed quite poorly in mammalian cells. This poorexpression cannot be overcome by use of a strong transcriptionalpromoter because the defects in the expression of intron-dependent genesare post-transcriptional in nature. Genomic versions of genes frequentlycannot be used because (i) they have yet to be isolated, or (ii) theyare too large to incorporate into useful expression vectors.

Many workers have tried to improve the expression of the cDNA versionsof genes by inserting an intron back into either the protein-codingregion of the gene or its 3' untranslated region. This approach isfrequently unsuccessful as well because many introns (i) cannot enableefficient processing and cytoplasmic accumulation of pre-mRNAs (e.g.,Ryu, 1989, supra; Nesic, et al., 1993, supra; Jonsson, et al., 1992,supra) or (ii) lead to the production of cryptically spliced mRNAs whichencode incorrect proteins (Huang, et al., Mol. Cell. Biol. 10:1805-1810,1990; Evans, et al., Gene 84:135-142, 1989).

Interestingly, although most pre-mRNAs in higher eukaryotes requireintrons for efficient mRNA biogenesis, this intron requirement is notuniversal. The genes encoding herpes simplex virus type 1 thymidinekinase (HSV-TK) (McKnight, Nucleic Acids Res. 8:5949-5964, 1980),histone proteins (Kedes, Annu. Rev. Biochem. 48:837-870, 1978),interferon-α (Nagata, et al., Nature 287:401-408, 1980), β-adrenergicreceptor (Koilka, et al., Nature 329:75-79, 1987), and c-jun (Hattori,et al., Proc. Natl. Acad. Sci. USA 9148-9152, 1988) are among thosegenes discovered to be naturally intronless yet expressed at functionallevels in higher eukaryotes.

To begin to understand the mechanism of intron-independent mRNAbiogenesis, Greenspan and Weissman (Mol. Cell. Biol. 5:1894-1900, 1985),Buchman and Berg (1988, supra), and Ryu (1989, supra) constructedplasmids in which an intron plus some adjacent exon sequence from anintron-requiring β-globin gene was placed 3' of the intronless sequencethat encodes HSV-TK. Greenspan and Weissman (1985, supra) found thatmuch of the resulting chimeric TK-globin RNA was polyadenylated andtransported to the cytoplasm without intron excision. All threelaboratories showed that the chimeric RNAs efficiently accumulated inmammalian cells regardless of whether an intron was present in theprimary transcript.

One hypothesis to explain these data is that transcripts synthesizedfrom β-globin and other intron-dependent genes contain negative,cis-acting RNA sequence elements that prevent them from being properlyprocessed and/or transported in the absence of introns; transcriptssynthesized from intron-independent genes lack these negative elementsand, thus, do not require introns for proper processing and transport.During the past decade, considerable data has accumulated in theliterature in support of this hypothesis. For example, Legrain andRosbash (1989, supra) found that mutations in splicing signals thatconverted an intron-containing gene into an intronless one led toefficient cytoplasmic accumulation of the intronless transcripts inyeast. Thus, they hypothesized that intronless transcripts aretransported to the cytoplasm by default pathways.

An alternative, non-mutually exclusive hypothesis is that transcriptssynthesized from intron-independent genes contain positive, cis-actingRNA sequence elements that enable them to be processed and transportedregardless of whether or not introns are present. Greenspan and Weissman(1985, supra) and Buchman and Berg (1988, supra) found that variousnon-overlapping regions of the HSV-TK gene accumulate in cells in theabsence of intron excision. Thus, HSV-TK transcripts are processed andtransported to the cytoplasm regardless of introns because they either(i) lack a negative cis-acting element, or (ii) contain multiple,positive, cis-acting elements. Their data could not distinguish betweenthese two hypotheses and was more supportive of the first hypothesis.

We show below that positive, cis-acting elements, called pre-mRNAprocessing enhancers (PPEs), exist. These sequence elements are capableof enabling intron-independent gene expression. Thus, theirincorporation into genes provides an alternative approach to insertingintrons for obtaining efficient processing and cytoplasmic accumulationof RNAs in higher eukaryotes.

SUMMARY OF THE INVENTION

To test whether transcripts of the HSV-TK gene actually do containpositive, cis-acting RNA sequence elements that enableintron-independent pre-mRNA processing and transport in highereukaryotes, we examined processing and transport in mammalian cells oftranscripts synthesized from an intronless variant of the human β-globingene containing insertions of various sequences from the HSV-TK gene. Wefound that a 119-nt sequence (SEQ ID NO:1) contained within thetranscribed region of the HSV-TK gene can enable efficient cytoplasmicaccumulation of β-globin transcripts in the absence of splicing.Furthermore, we also found that hnRNP L, an abundant, 68 kDa cellularprotein of previously unknown function, associates sequence-specificallywith this pre-mRNA processing enhancer (PPE), but not with a mutantvariant of it defective in rescuing the cytoplasmic accumulation ofintronless human β-globin transcripts. The intronless cellular genec-jun was also found to contain sequences that enable intron-independentpre-mRNA processing and transport. Thus, intron-independent pre-mRNAprocessing and transport probably involves sequence-specific RNA-proteininteractions between PPEs and appropriate cellular factors such as hnRNPL.

In one embodiment, the present invention is a chimeric RNA moleculecontaining one or more pre-mRNA processing enhancers. The chimeric RNAmolecule comprises a first and a second RNA sequence. The first RNAsequence contains a pre-mRNA processing enhancer that is not nativelyconnected to the second RNA sequence.

Preferably, the RNA comprises a 5' untranslated region, a 3'untranslated region, and a protein-encoding region. Inserted into the 5'untranslated region or 3' untranslated region is at least one PPEsequence. The PPE sequence is not natively connected to theprotein-encoding sequence.

Preferably, the chimeric RNA molecule comprises two or more PPEsequences.

In another embodiment, the present invention is an isolated populationof DNA molecules containing one or more pre-mRNA processing enhancers(PPEs). These molecules are intended to be used as "cassettes" thatwould be inserted into genetic constructs to enhance the expression ofintronless RNA molecules that would otherwise accumulate at anunsatisfactory level. For example, the cassette may be placed 5' or"upstream" of a intronless version of a protein-encoding sequence. Sucha sequence is typically created during cDNA cloning. Preferably, the PPEwould be placed 3' or "downstream" of an operable transcriptionalpromoter sequence.

In another embodiment, the present invention is a gene constructcomprising at least one (preferably two) pre-mRNA processing enhancersdownstream of a transcriptional promoter and 5' of a restrictionendonuclease site and signals for transcription termination and 3'-endformation (e.g., polyadenylation signal, ribozyme cleavage signal). Thisrestriction endonuclease site is preferably unique in the vector anddesigned to accommodate a protein-encoding sequence. Such a constructwould be useful to allow one to place an RNA-encoding sequence of choicedownstream of a PPE.

In another embodiment, the present invention is a method to enhancecytoplasmic accumulation of intronless RNAs comprising the steps ofinserting an RNA-encoding DNA sequence into the vector described aboveand expressing the RNA sequence (e.g., to enable efficient cytoplasmicaccumulation of "anti-sense" RNAs).

In another embodiment, the present invention is a kit for enhancing thecytoplasmic accumulation of intronless RNAs comprising the vectordescribed above.

In another embodiment, the present invention is a gene constructcomprising at least one pre-mRNA processing enhancer operably connectedto a DNA sequence intended to be expressed as an RNA molecule. The geneconstruct is designed so that the PPE is expressed within the 5'untranslated region of the RNA expression product.

It is an object of the present invention to provide a DNA sequencecapable of being inserted into a genetic construct so that the DNAsequence is expressed as part of the 5' untranslated region or 3'untranslated region of an RNA molecule. Thus, the DNA sequence willenable one to form an mRNA product with enhanced cytoplasmicaccumulation.

It is another object of the present invention to provide SEQ ID NO:1, anexample of a PPE.

It is another object of the present invention to provide a geneconstruct suitable for enhancing the cytoplasmic accumulation of RNAmolecules.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and figures.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C demonstrate that the presence of HSV-TK sequences in cis canobviate the intron requirement for processing of human β-globintranscripts. FIG. 1A shows the structures of plasmids containing aninsertion of the transcribed region of the HSV-TK gene into the5'-untranslated region of the human β-globin gene and a summary of thedata obtained with these plasmids. (Noteworthy is our finding thatinsertion of the HSV-TK sequences into the β-globin gene increasescytoplasmic accumulation of intronless transcripts at least 45-fold from<0.01 to 0.45.) FIG. 1B is a schematic diagram of the human β-globinprobe used in the S1 nuclease mapping analysis and the size of the DNAfragments resulting from protection by hybridization with thecorresponding RNA. FIG. 1C is a schematic diagram of the cellularβ-actin probe used as an internal control in the S1 nuclease mappingexperiment.

FIG. 2 is a diagram of the structure of various plasmids containingdifferent portions of the HSV-TK gene inserted into the NcoI site ofhuman β-globin gene and the data obtained with these plasmids. The NcoIsite is situated in the 5' untranslated region of this gene. Theinserted HSV-TK sequences are relative to the transcription initiationsite in the HSV-TK gene (McKnight, et al., Cell 25:385-398, 1981). Thedata in the column on the right are the amount of β-globin-like RNAaccumulated in the cytoplasm of cells transfected with the plasmidcontaining the cDNA version of the indicated gene relative to the amountaccumulated in cells transfected in parallel with the plasmid containingthe corresponding genomic version of this gene. To determine the effectof the insertion of a given TK sequence on intronless globin RNAaccumulation, the numbers in the right column should be compared to eachother. For example, insertion of the TK119 sequence into the NcoI siteof the cDNA version of the human β-globin gene increases cytoplasmicaccumulation of β-globin RNA at least 20-fold (i.e., 0.20 vs. less than0.01).

FIG. 3 is a diagram of the structures of plasmids containing aninsertion of the HSV-TK element in the sense vs. anti-sense orientation.

FIG. 4 is a diagram of plasmids containing tandem copies of the HSV-TKnt 361-to-479 and the data obtained with these plasmids.

FIG. 5 is a diagram of mutations in the HSV-TK PPE and the abilities ofthese variants to enable intron-independent gene expression (SEQ ID NOS.6-9).

FIG. 6 is a set of schematic diagrams of the transcribed regions of theplasmids with linker-scanning mutations in the PPE used to synthesizethe radiolabeled rTK119, rTK119LSO, rTK119LS1, and rTK119LS2 RNAs andthe summary of the UV-crosslinking data obtained with these RNAs.

FIG. 7 describes the structures of plasmids containing an insertion ofpart of the coding region of the c-jun gene into the 5' untranslatedregion of the human β-globin gene and a summary of the data obtainedwith these plasmids.

DESCRIPTION OF THE INVENTION 1. In General

One problem frequently encountered in trying to efficiently expressgenes in mammalian cells in culture or animals is very poor expressionof the cDNA version of the gene of interest. The cDNA version of an mRNAplaced downstream of a transcriptional promoter is one example of an"intronless gene." We refer to a transcript or pre-mRNA synthesized fromsuch an intronless gene as an "intronless" transcript or pre-mRNA. Afterprocessing and transport to the cytoplasm, these RNAs are referred to as"intronless" RNAs or mRNAs.

We have identified novel sequence elements, called pre-mRNA processingenhancers or "PPEs", that enable efficient cytoplasmic accumulation ofintronless RNAs in the absence of splicing. The inclusion of PPEs inexpression vectors will enable one to readily achieve efficientexpression of intron-dependent genes in the absence of splicing for usein (i) gene therapy, (ii) the manufacture of proteins, and (iii) basicand applied research. For example, the inclusion of a PPE in anexpression vector would enable one to express an mRNA encoding atherapeutic protein in a gene therapy application where expression of asufficient amount of the therapeutic protein is vital to the success ofthe treatment.

The present invention involves the use of PPEs in enhancing cytoplasmicaccumulation of mRNAs. In one embodiment, the present invention is anmRNA molecule comprising a first and a second RNA sequence. The firstRNA sequence comprises at least one PPE. The PPE sequence is notnatively connected to the second RNA sequence.

Preferably, the chimeric RNA comprises a 5' untranslated region, a 3'untranslated region, and a protein- encoding sequence. The chimeric RNAcomprises at least one PPE within the 5' untranslated region or the 3'untranslated region of the mRNA molecule. The PPE is not nativelyassociated with the protein-encoding sequence or with any other part ofthe 5' untranslated region or the 3' untranslated region containing thePPE. Most preferably, the PPE is present in two copies.

As described more fully below, the present invention is also a geneconstruct designed to enhance cytoplasmic accumulation of intronlessRNAs or mRNAs and a method for enhancing cytoplasmic accumulation of anintronless RNA or mRNA that utilizes this gene construct.

In another embodiment, the present invention is a preparation of DNAmolecules comprising at least one PPE. These molecules would act as a"cassette" to be inserted into a DNA construct 5' of an RNA-encodingsequence. Preferably, the PPE would be placed downstream from afunctional promoter.

2. Suitable PPEs

In accordance with the present invention, we have demonstrated thepresence and isolation of a PPE sequence in at least two differentgenes. First, the examples below disclose that the transcribed region ofthe naturally intronless HSV-TK gene contains at least one positive,cis-acting sequence element that can enable the proper processing andtransport of transcripts synthesized from intronless variants ofintron-dependent β-globin genes. We have: (i) localized to a 119-bpregion a sequence that can mediate this effect (SEQ ID NO:1, FIG. 2),(ii) shown that this novel sequence element truly enables cytoplasmicaccumulation of mRNAs in the absence of splicing (Liu and Mertz, Genes &Dev., in press), (iii) provided evidence that this sequence functions atthe RNA level (FIGS. 3 and 6), (iv) demonstrated that the sequencefunctions in an orientation-dependent manner (FIG. 3), and (v) shownthat this element functions more efficiently when present in more thanone copy (FIG. 4). We name sequence elements with this novel set ofproperties PPEs for pre-mRNA processing enhancers.

While SEQ ID NO:1 is a preferred PPE of the present invention, weenvision that modifications of SEQ ID NO:1 are also suitable PPEs. It iswell known in the art of molecular biology that minor nucleotidechanges, additions or deletions may result in no change in thefunctional nature of the nucleotide sequence. For example, changes innucleotide 338 (G→A) and 333 (C→G) were tested and did not alteractivity of this element. Any nucleotide change or addition that retainsat least 25% of the PPE activity of SEQ ID NO:1 is a suitable PPE of thepresent invention.

For example, FIG. 5 demonstrates some alterations made to the original119 bp PPE. The sequence alterations present in TK119LSO inactivate thiselement as a PPE. On the other hand, the sequence alterations present inTK119LS2 enable this sequence to function as a PPE at approximately 65%the level of TK119. Thus, it is still a suitable, thoughless-than-optional, PPE. Similarly, some deletions, insertions, orsubstitution mutations in the sequence would probably result in asuitable PPE. TK119LS1 is also a suitable PPE at approximately 30% ofthe activity of TK119.

Second, we also show that sequences contained within the naturallyintronless cellular gene c-jun can also provide this function (FIG. 7).Thus, at least some cellular genes also contain PPEs and more than onesequence can function as a PPE.

A PPE of the present invention is isolated from the majority of thesurrounding gene. Therefore, a 300 bp or larger naturally occurringnucleotide segment containing a single PPE is not a PPE of the presentinvention. Preferably, a single PPE is localized to a nucleotidesequence less than 200 bp, most preferably less than 140 bp. A PPE must(1) be localized to a nucleotide length of less than 300 bp, (2)function in an orientation-dependent manner, (3) function moreefficiently when present in more than one copy, and (4) enhance thecytoplasmic accumulation of an intronless (i.e., cDNA) version of anintron-dependent transcript by at least 2-fold, preferably by at least5-fold (20-fold or more for β-globin).

Insertion of most of the transcribed region of the HSV-TK gene can, inlarge part, relieve the requirement for an intron for efficientcytoplasmic accumulation of intronless human β-globin transcripts (Ryu,1989, supra; FIG. 1). Our deletion mapping data (FIG. 2) indicated thatan element contained within nt 361-to-479 (relative to the transcriptioninitiation site) of the HSV-TK gene can largely provide this function.Greenspan and Weissman (1985, supra) and Buchman and Berg (1988, supra)had noted previously that non-overlapping sequences transcribed from theHSV-TK gene can accumulate in mammalian cells in the absence of intronexcision. The simplest interpretation of their data was that intronlessHSV-TK transcripts lacked negative, cis-acting sequence elements thatprevent nuclear export in the absence of splicing. In view of thefindings presented here, we now reinterpret their data to indicate,instead, that the HSV-TK gene probably contains at least two PPEs.Consistent with this conclusion are our observations that (i) the HSV-TKPPE we mapped here functions with approximately one-half the efficiencythat the full-length HSV-TK gene does (FIG. 1 vs. FIG. 2), and (ii)insertion of two copies of this PPE results in a two-fold increase inthe cytoplasmic accumulation of intronless transcripts (FIG. 3). Thus,we hypothesize that multiple copies of PPEs may act in a cooperative oradditive manner to enhance the efficiency of processing and nuclearexport of pre-mRNAs.

In accordance with the present invention, one may wish to isolate a PPEsequence from an intronless gene other than c-jun or HSV-TK. One wouldfirst examine the transcribed region of the selected gene, as we havedone below with the HSV-TK gene. Deletion analysis as demonstrated belowwill enable one to find the appropriate PPE sequence. As describedabove, the PPE must be isolated from the surrounding gene sequence. Asequence is a suitable PPE sequence if the sequence (1) enhancescytoplasmic accumulation of an intronless transcript by at least 2-fold,(2) functions more efficiently when present in more than one copy, (3)functions in an orientation-dependent manner, and (4) is less than 200nucleotides in length. Preferably, the PPE enhances cytoplasmicaccumulation of an intronless RNA at least 5-fold. Most preferably, theincrease is at least 20-fold. We note that if two PPE sequences arepresent, the combined length may be greater than 200 nucleotides.

3. Suitable RNA Molecules

Pre-mRNAs differ considerably in their requirement for an intron forefficient processing. Introns are absolutely required for expression ofthe human β-globin (Ryu, 1989, supra; Collis, et al. 1990, supra),rabbit β-globin (Buckman and Berg, 1988, supra), human PNP (Jonsson, etal., 1992, supra) and human TPI (Nesic, et al., 1993, supra) genes. Onthe other hand, some naturally intron-containing genes i.e., thoseencoding polyoma middle T antigen (Treisman, et al., Nature 292:595-600,1981) and cellular thymidine kinase (Gross, et al., Mol. Cell. Biol.7:4576-4581, 1987)! appear to be considerably less dependent upon thepresence of introns for processing of their transcripts. Nevertheless,the presence of introns still increases the cytoplasmic accumulation ofthese latter RNAs at least a few fold.

We envision the present invention to be useful for a variety of mRNAs.Additionally, the present invention is envisioned to be useful inexpressing RNA molecules that are not necessarily translated into aprotein product. For example, ribozymes and other catalytic RNAs mightbe usefully expressed by the present invention. Additionally, anti-senseRNAs would also be advantageously expressed.

4. Gene Constructs

In another embodiment, the present invention is a gene constructcomprising a DNA sequence encoding at least one copy of a PPE,preferably 5' of a unique restriction endonuclease site. This vector isconstructed to accommodate the insertion of an RNA-encoding sequencewhose transcript would be accumulated with the addition of a PPE.Typically, the PPE is downstream from a promoter sequence. Preferably,more than one PPE is present in the gene construct. In an especiallypreferred embodiment, the restriction site is within a polylinkercomposed of multiple unique restriction endonuclease sites. In anespecially preferred embodiment, the construct additionally contains atranslation enhancer, such as the enhancer described in U.S. Pat. No.4,937,190 to Palmenberg, et al. This enhancer would typically be placeddownstream of the PPE and upstream of a protein-encoding sequence.

The gene construct of the present invention may be contained within avariety of vectors. Preferential examples are a virus or plasmid vector.

The present invention is also a method of enhancing the cytoplasmicaccumulation of RNA molecules. The method comprises the step ofinsertion of an RNA-encoding sequence into the vector described aboveand expressing that RNA sequence.

The present invention is also a gene construct comprising at least onecopy of a PPE downstream from a functional promoter and upstream from aprotein-encoding sequence. Such a gene construct could be useful in manyways. For example, one might make a library of random cDNA sequenceswithin plasmid vectors. These plasmid vectors could contain the PPEsequence in the orientation and location described above. This librarywould then be suitable for transfection into or transformation ofappropriate host cells, such as mammalian cells. Expression of the RNAsand encoded proteins would be at a level sufficient to screen for thedesired cDNA.

5. Kits for Enhancing RNA Accumulation

The present invention is also a kit for the enhanced cytoplasmicaccumulation of RNAs comprising the restriction endonucleasesite-containing vector described above. The kit would typically containa receptacle containing the restriction endonuclease site-containingvector described above. This vector would be suitable for a kit user toinsert an intronless RNA-encoding DNA of interest and, subsequently,obtain efficient cytoplasmic accumulation of the RNA and, whereappropriate, translation of the mRNA in a biological system.

The present invention is also a kit for assaying with very highsensitivity for gene expression in single or small numbers of cells. Forexample, the kit might contain a reporter construct containing a PPEupstream of reporter gene sequences, such as luciferase-encoding orβ-galactosidase-encoding sequences. When expressed from a specificinserted transcriptional enhancer/promoter sequence in some cells of achimeric or transgenic animal, the expressing cells could be detectedwith improved sensitivity because of the increased abundance of thereporter messages.

EXAMPLES 1. In General

Most pre-mRNAs require an intron for efficient processing in highereukaryotes. Exceptions to this rule are transcripts synthesized fromnaturally intronless genes. To test the hypothesis thatintron-independent gene expression involves positive, cis-acting RNAsequence elements, we constructed chimeric genes in which variousregions of the HSV-TK gene were inserted into an intronless variant ofthe highly intron-dependent human β-globin gene. Plasmids containingthese chimeric genes were transfected into CV-1PD cells. The structuresand quantities of the resulting globin-like RNAs were determined by S1nuclease mapping. A 119-nt sequence element (SEQ ID NO:1) containedwithin nucleotide residues 361-479 relative to the transcriptioninitiation site of the HSV-TK gene (McKnight, et al., 1981, supra) wasfound to enable efficient cytoplasmic accumulation of globin-like RNA inthe absence of splicing in an orientation-dependent manner. RNAUV-crosslinking assays indicated that a 68 kDa protein present innuclear extracts of HeLa and COS cells binds specifically to thispre-mRNA processing enhancer (PPE). Analysis of substitution mutants inthis PPE indicated that binding of the 68 kDa protein correlates withaccumulation of the chimeric RNA in the cytoplasm. A sequence from thetranscribed region of the intronless cellular gene c-jun was also foundto enable efficient processing of intronless β-globin-like transcripts.Thus, we conclude that: (i) intronless genes contain PPEs, (ii) the 68kDa protein is a sequence-specific RNA-binding protein, and (iii)intron-independent pre-mRNA processing and transport may involvesequence-specific RNA-protein interactions between PPEs and proteinssuch as the 68 kDa protein. We propose that PPEs may be of general usefor the efficient expression of cDNA versions of intron-dependent genes.

2. Materials and Methods

Cells, Transfections, and Nuclear Extracts

The African green monkey kidney cell line CV-1PD was grown in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 5% fetal bovine serumas described previously (Good, et al., J. Virol. 62:563-571, 1988).Co-transfections were performed by a modification of theDEAE-dextran/chloroquine procedure essentially as described previously(Liu and Mertz, 1993, supra). The relative transfection efficiencieswere determined as described previously (Ryu and Mertz, 1989, supra) bySouthern blot analysis of the replicated plasmid DNA present in eachsample. HeLa and COS cell nuclear extracts were prepared essentially asdescribed previously (Dignam, et al., Nucleic Acids Res. 11:1475-1488,1983; Terns, "Role of nuclear poly(A) polymerase in the 3'-endprocessing of precursor messenger RNA," Ph.D. Thesis. Pennsylvania StateUniversity, State College, Pennsylvania, 1990).

Recombinant Plasmids

All plasmids were constructed by standard recombinant DNA techniques(Sambrook, et al., Molecular Cloning: A Laboratory Manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Plasmidpβ1(+)2(+) contains a genomic version of the human β-globin gene (FIG.1). Plasmid pβ1(-)2(-) is identical in sequence to pβ1(+)2(+) except forthe precise lack of the two β-globin introns. These plasmids have beendescribed in detail elsewhere (Ryu, 1989, supra; Yu, et al., 1991,supra). Plasmids pTKβ1(-)2(-) and pTKβ1(+)2(+) contain an insertion ofnucleotide residues (nt) 59-to-1238 (relative to the transcriptioninitiation site) of the HSV-TK gene into the NcoI site in the5'-untranslated region of the plasmids pβ1(-)2(-) and pβ1(+)2(+),respectively (FIG. 1). These plasmids were constructed by ligation afterthe HSV-TK sequence to be inserted was generated by PCR-basedamplification as described previously (Liu, 1994, supra.). Similarstrategies were used in construction of pJunβ1(-)2(-) and pJunβ1(+)2(+)in which the entire coding region except the terminal codon of c-jun wasinserted. A series of TK-deleted, duplicated, and anti-sense-insertedvariants of the TK-globin chimeric plasmids (FIGS. 2, 3, and 4; Table 1)were generated likewise (Liu, 1994, supra; Liu and Mertz, 1995, inpress, Genes and Development). The construction of pTK119Xβ1(-)2(-) andpTK119Xβ1(+)2(+) and the linker-scanning mutant derivatives of theseplasmids (FIGS. 5 and 6; Table 1) has also been described in detailelsewhere (Liu, 1994, supra; Liu and Mertz, 1995, supra).

Plasmid pT7/TK119 (FIG. 6) was constructed by insertion of the 119-bpBspHI and NcoI-digested PCR fragment of pTK119β1(-)2(-) intoNcoI-digested pGEM5Z(+) in the sense orientation. Plasmids pT7/TK119LS0,pT7/TK119LS1, and pT7/TK119LS2 are identical to pT7/TK119 except for theindicated substitution mutations in the TK sequence element (Table 1;FIG. 6) and some extra globin sequences in the polylinker region 3' ofthe TK sequences. The RNAs synthesized from these plasmids aredesignated by "rTK" followed by the length of the RNA and, whereappropriate, the mutation name. Plasmids pT7/TK119β1(-)2(-) andpT7/β1(-)2(-) were constructed by insertion of the HindIII- andXbaI-digested PCR fragments of pTK119β1(-)2(-) and pβ1(-)2(-),corresponding to the sequences from the transcription initiation site to91 bases 3' of the human β-globin polyadenylation signal, into HindIII-and XbaI-digested pGEM2 vector DNA (Promega). Plasmids pJunβ1(+)2(+) andpJunβ1(-)2(-) contain an insertion of nt 975-to-1963 (relative totranscription initiation site) of the c-jun gene (Hattori, et al., 1988,supra) into the NcoI site in the 5'-untranslated region of the plasmidspβ1(+)2(+) and pβ1(-)2(-), respectively; these plasmids were constructedby PCR amplification and insertion as described above.

RNA Purification and S1 Nuclease Mapping Analysis

Nuclear and cytoplasmic RNAs were purified from monkey cells 48 hoursafter transfection as described previously (Liu and Mertz, 1993, supra).The relative amounts of globin-like RNA accumulated in the nucleus andcytoplasm were determined by quantitative S1 nuclease mapping techniquesas described previously (Liu and Mertz, 1993, supra). The probes used inthe S1 nuclease mapping analyses are shown in the figures. Cellularβ-actin RNA, mapped concurrently, served as an internal control forrecovery of the RNA samples and purity of the nuclear RNA (Yu, et al.,1991, supra; Liu and Mertz, 1993, supra). Southern blot analysis of therelative amount of DpnI-resistant, β-globin-encoding plasmid DNA presentin each nuclear sample prior to treatment with DNase I was performed asdescribed elsewhere (Ryu, 1989, supra; Liu and Mertz, 1993, supra); itwas used to assay for both (i) nuclear contamination of cytoplasmicnucleic acid, and (ii) differences in transfection efficiencies (datanot shown). The S1 nuclease-protected DNA fragments were electrophoresedin 8M urea, 5% polyacrylamide gels. Quantitations were performed byscanning with a PhosphorImager (Molecular Dynamics).

RT-PCR, PCR, and DNA Sequencing Analyses

Prior to reverse transcription, each RNA sample for RT-PCR analysis wastreated with RNase-free FPLC-pure DNase I (Pharmacia). Each reversetranscription reaction contained 2% of the cytoplasmic RNA harvestedfrom a 100-mm dish of transfected cells, 25 units AMV reversetranscriptase (Boehringer Mannheim), 25 ng 3'-anti-sense primer(5'-TTAGGCAGAATCCAGATGCTCAAGGCC-3', SEQ ID NO:2), 1.25 mM of each of thefour dNTPs, and 20 units RNasin (Promega) in a total volume 20 μl. Afterincubation at 42° C. for 1.5 hours, the reaction mixture was incubatedat 95° C. for 10 minutes and quickly chilled on ice to denature theheteroduplexes. Afterward, the PCR reaction was performed in a 50 μlvolume containing 10 mM Tris-HCl (pH 8.3), 1.5 mM Mg2Cl, 50 mM KCl, 1.25mM of each of the four dNTPs, 2.5 unit Taq polymerase, 1 μM each of the3'-antisense primer and the 5' primer (5'-ACATTTGCTTCTGACACAACTGTG-3',SEQ ID NO:3), and the 20 μl from the reverse transcription reaction. APerkin-Elmer Cetus thermal cycler was used with denaturation at 94° C.for 1 minute, primer annealing at 55° C. for 1 minute, and extension at72° C. for 2 minutes for 35 cycles.

UV-crosslinking Assays

The RNA substrates for in vitro studies were synthesized using acommercial T7/SP6 in vitro transcription kit (Promega). The DNAs used astemplates for the RNA syntheses were derivatives of pGEM5Z(+) (Promega).One microgram of each template was linearized by digestion with NcoI andtranscribed with T7 RNA polymerase at 37° C. for 1 hour in the presenceof either α-³² P!UTP or α-³² P!CTP (3000 Ci/mmol, Amersham). Wild-typeand mutant RNA substrates were prepared in parallel to insure theirspecific activities were similar. After RNA synthesis, the DNA templateswere degraded by incubation with RNase-free DNase I (Promega) for 15minutes at 30° C., and the full-length, radiolabeled transcripts werepurified by polyacrylamide gel electrophoresis as described previously(Liu, 1994, supra). For UV-crosslinking assays, 5×10⁴ cpm of ³²P-labeled RNA was incubated with 5-10 μg HeLa or COS cell nuclearextract, 10 mM KCl, 10% glycerol, 0.2 mM dithiothreitol, and 2 μg yeasttRNA (Boehringer Mannheim) in a total volume of 10 μl at 30° C. for 10minutes (Gillis and Malter, J. Biol. Chem. 266:3172-3177, 1991).Afterward, the reaction mixture was irradiated in a UV-Stratalinker(Stratagene) for 10 minutes on the automatic setting. RNase A was addedto a final concentration of 1 mg/ml, and incubation was continued at 37°C. for 15 minutes. After addition of SDS-PAGE sample buffer, each samplewas incubated at 100° C. for 4 minutes and electrophoresed in a 15%polyacrylamide gel containing 0.1% SDS. The gels were either dried andautoradiographed overnight at -70° C. or exposed to a PhosphorImagerscreen and scanned in a PhosphorImager (Molecular Dynamics). CompetitionUV-crosslinking assays were performed similarly, except forpre-incubation of the nuclear extract with unlabeled RNA preparedfollowing the protocol of Gurevich, et al. (1991).

3. Sequences Contained Within the Transcribed Region of the HSV-TK GeneEnable Efficient Processing of Intronless Human β-globin Transcripts

We first constructed pTKβ1(-)2(-) and pTKβ1(+)2(+), plasmids into whichthe HSV-TK nucleotide residues 59-to-1238 (relative to the transcriptioninitiation site) were inserted in the sense orientation into a completecDNA and genomic version, respectively, of the human β-globin gene (FIG.1A). The genomic version of the hybrid gene served as a control formessage stability in the cytoplasm. Since the mature mRNAs generatedfrom these plasmids are identical in primary structure, they should haveidentical half-lives in the cytoplasm unless processing via alternativepathways in the nucleus affects the location or association withribonuclear proteins in the cytoplasm. Therefore, we assumed that theratio of TK-globin chimeric RNA accumulated in the cytoplasm of cellstransfected in parallel with the cDNA relative to the genomic version ofa hybrid gene provides a reasonable indication as to the effectivenessof the HSV-TK sequence in allowing intron-independent gene expression.

These plasmids were co-transfected in parallel into monkey cells alongwith pRSV-Tori, a plasmid encoding the SV40 large T antigen (Ryu, 1989,supra; Yu, et al., Nucleic Acids Res. 19: 7231-7234, 1991). The presenceof the latter plasmid results in replication of the test plasmid to highcopy number, making structural and quantitative analysis of theaccumulated β-globin-like RNAs easy to perform by quantitative S1nuclease mapping techniques (Ryu, 1989, supra; Yu, et al., 1991, supra;Liu and Mertz, Nucleic Acids Res. 21:5256-5263, 1993; Ryu, et al.,1995). As negative and positive controls, we also transfected inparallel the plasmids pβ1(-)2(-) and pβ1(+)2(+) which contain cDNA andgenomic versions of the human β-globin gene, respectively. The amountsof cytoplasmic and nuclear globin-like RNA accumulated in cellstransfected with each plasmid were determined relative to the amountsaccumulated in pβ1(+)2(+)-transfected cells.

FIG. 1 demonstrates that the presence of HSV-TK sequences in cis canobviate the intron requirement for processing of human β-globintranscripts. FIG. 1A shows the structures of plasmids containing aninsertion of the transcribed region of the HSV-TK gene into the5'-untranslated region of the human β-globin gene and a summary of thedata obtained with these plasmids. Only the transcribed region of eachgene is shown. The remainder of each plasmid is identical in sequenceand described in detail elsewhere (Ryu, 1989, supra; Yu, et al., 1991,supra). Referring to FIG. 1A, shaded boxes indicate human β-globin exonsequences; open boxes indicate human β-globin intron sequences; hatchedboxes indicate sequences from the transcribed region of the herpessimplex virus type 1 thymidine kinase (HSV-TK) gene; numbers at ends ofhatched boxes indicate endpoints of the HSV-TK sequence inserted in ntrelative to the HSV-TK gene's transcription initiation site; B indicatesBamHI; N indicates NcoI; E indicates EcoRI. The first column on theright indicates the amount of β-globin-like RNA present in the nucleusof cells transfected with each plasmid relative to the amountaccumulated in cells transfected in parallel with pβ1(+)2(+). The secondcolumn indicates the amount of β-globin-like RNA accumulated in thecytoplasm of these same cells relative to that accumulated in thepβ1(+)2(+)-transfected cells. The last column on the right indicates theamount of β-globin-like RNA present in the cytoplasm of cellstransfected with the plasmid containing the cDNA version of the generelative to the amount accumulated in cells transfected in parallel withthe plasmid containing the corresponding genomic version of this gene,with normalization to the relative amounts of (i) cellular β-actinpresent in the same RNA samples, and (ii) replicated β-globin-encodingplasmid DNA present in the nuclear samples obtained from these cells(data not shown). These data are means ±S.E.M.s from two experiments andwere obtained from electrophoretic gels.

FIG. 1B is a schematic diagram of the human β-globin probe (describedpreviously by Ryu, 1989, supra; Yu, et al., 1991, supra) used in the S1nuclease mapping analysis and the sized DNA fragments resulting fromprotection by hybridization with the corresponding RNAs. The humanβ-globin probe was 5' end-labeled at the BamHI site; the wavy lineindicates the discontinuity between the probe and the globin RNA.Abbreviations are as described in panel A.

FIG. 1C is a schematic diagram of the cellular β-actin probe, describedpreviously (Ryu, 1989, supra; Yu, et al., 1991, supra), used as aninternal control in the S1 nuclease mapping experiment. The actin probewas 5' end-labeled at the RsaI site. This probe has pBR322 sequences,indicated by wavy line, adjacent to the SalI site of the β-actin gene.

Referring to FIG. 1, as expected, little, if any RNA synthesized fromthe cDNA version of the gene was detectable in the cytoplasm. On theother hand, RNA synthesized from the genomic version accumulated to highlevels. Insertion of sequences from the HSV-TK gene into the5'-untranslated region of the human β-globin cDNA also enabled highlevel accumulation of globin-like RNA in the cytoplasm. In sharpcontrast to the greater than 100-fold difference in cytoplasmicaccumulation observed between cells transfected with pβ1(-)2(-) versuspβ1(+)2(+), only a 2- to 3-fold difference was observed between cellstransfected with pTKβ1(-)2(-) versus pTKβ1(+)2(+). Therefore, sequencescontained within the transcribed region of the HSV-TK gene can enableefficient processing and cytoplasmic accumulation of intronless chimericTK-globin RNAs.

4. Localization of an HSV-TK Sequence Element MediatingIntron-independent Gene Expression

To look for a positive, cis-acting sequence element in the HSV-TK genethat might enable intron-independent expression of the human β-globingene, we made a series of TK-deleted variants of pTKβ1(-)2(-) andpTKβ1(+)2(+) (Table 1, below; FIG. 2). FIG. 2 demonstrates that regionsof the HSV-TK gene differ in their ability to enable intron-independentexpression of the human β-globin gene. Specifically, FIG. 2 is a summaryof the structures of plasmids containing different portions of theHSV-TK gene inserted into the NcoI site of the human β-globin gene andthe data obtained with these plasmids. The schematic diagrams indicatethe regions of the HSV-TK gene inserted into the cDNA and genomicversions of the human β-globin gene as described in Table 1. The symbolsare the same as those described in the legend to FIG. 1. Only the TKpart of the name of each plasmid is stated.

Still referring to FIG. 2, the column on the right indicates the amountof TK-globin chimeric RNA accumulated in the cytoplasm of cellstransfected in parallel with the cDNA versus corresponding genomicversion of each pair of plasmids.

Each of these plasmids contains a portion of the transcribed region ofthe HSV-TK gene inserted at the NcoI site in the 5'-untranslated regionof either the cDNA or genomic version of the human β-globin. Therelative amounts of the TK-globin chimeric RNAs accumulated in cellstransfected with each of these plasmids were determined as describedabove (summarized in FIG. 2).

Table 1 below summarizes the structures of the TK-globin chimeric genesexamined herein.

                  TABLE 1                                                         ______________________________________                                                     HSV-TK nucleotide residues inserted                              Plasmid.sup.a                                                                              (altered) into globin NcoI site                                  ______________________________________                                        pβ1(+)2(+).sup.b                                                                      --                                                               pβ1(-)2(-).sup.c                                                                      --                                                               pTKβ1(-)2(-)                                                                          59-1238                                                          pTK583β1(-)2(-)                                                                       170-752                                                          pTK415β1(-)2(-)                                                                       338-752                                                          pTK311β1(-)2(-)                                                                       442-752                                                          pTK142β1(-)2(-)                                                                       338-479                                                          pTK119β1(-)2(-)                                                                       361-479                                                          pTK69β1(-)2(-)                                                                        411-479                                                          pTK38β1(-)2(-)                                                                        338-479 (delete 341-444)                                         pTK119ASβ1(-)2(-)                                                                     361-479 antisense                                                pTK119Xβ1(-)2(-)                                                                      361-479 (438 G→A; 433 C→G)                         p2XTK119β1(-)2(-)                                                                     361-479:361-479 (438 G→A; 433 C→G)                 pTK119LS0β1(-)2(-)                                                                    361-479 (438 G→A; 433 C→G; 407-419                              ATCTACACCACA (SEQ ID NO:4)→                                            TAGTAGATCTAGA (SEQ ID NO:5)                                      pTK119LS1β1(-)2(-)                                                                    361-479 (438 G→A; 433 C→G; 416-421                              ACACAA→AGATCT).sup.d                                      pTK119LS2β1(-)2(-)                                                                    361-479 (438 G→A; 433 C→G; 422-427                              CACCGC→AGATCT).sup.d                                      ______________________________________                                         .sup.a Only the cDNA version of each plasmid is shown; the genomic versio     of each variant, containing IVS1 and IVS2, was made likewise.                 .sup.b pβ1(+)2(+) is the parental starting plasmid; it contains          nucleotide residues -812 through +2156 of the human g1obin gene, includin     IVS1 and IVS2.                                                                .sup.c pβ1(-)2(-) is identical to pβ(+)2(+) except for the          precise deletion of IVS1 and IVS2.                                            .sup.d A unique Bg1II site (5AGATCT-3') was introduced into each plasmid      during the mutagenesis.                                                  

Different HSV-TK sequences were found to differ significantly in theirability to enable cytoplasmic accumulation of globin-like RNA in theabsence of introns. For example, whereas the presence of HSV-TK nt338-to-752 increased cytoplasmic accumulation of globin-like RNA fromintronless transcripts at least 30-fold, the presence of HSV-TK nt442-to-752 had little effect. Thus, a sequence contained, at least inpart, within HSV-TK nt 338-to-442 can provide in cis an elementnecessary for efficient pre-mRNA processing.

To delineate further the region of the HSV-TK gene that contains thiselement, we constructed additional plasmids containing insertions ofsmaller and smaller portions of the nt 338-to-752 region of the HSV-TKgene. Analysis of the globin-like RNAs accumulated in cells transfectedwith these plasmids indicated that the presence of HSV-TK nt 361-to-479is sufficient to enable significant cytoplasmic accumulation ofglobin-like RNA. However, the presence of nt 411-to-479 is notsufficient. Therefore, nt 361-to-479 of the HSV-TK gene contains apositive, cis-acting sequence element that enables intron-independentprocessing of β-globin transcripts, with at least part of this elementbeing contained within nt 361-to-410. We name sequence elements withthis function PPEs for pre-mRNA processing enhancers.

5. HSV-TK PPE Mediates Cytoplasmic Accumulation of β-globin RNA in theAbsence of Splicing

One trivial possibility is that the TK-globin chimeric RNA accumulatedin the cytoplasm of cells transfected with pTK119β1(-)2(-) because thepresence of cryptic splice sites enabled splicing to occur despite theabsence of known introns. To test this hypothesis, we performed anadditional structural analysis of the chimeric RNA accumulated in thecytoplasm using a reverse transcriptase-polymerase chain reaction(RT-PCR) assay with primers corresponding to sequences near the 5'- and3'-ends of the RNA. All of the TK-globin chimeric RNAs accumulated incells transfected with the intronless plasmid pTK119β1(-)2(-) weresimilar in size to the processed TK-globin chimeric RNAs accumulated incells transfected with the intron-containing plasmid pTK119β1(+)2(+)(data not shown). No bands corresponding to cryptically spliced productswere detected. Neither were discontinuities in the RNA detected by S1nuclease mapping with a probe homologous to pTK119β1(-)2(-) (data notshown). Therefore, we conclude that the PPE contained within nt361-to-479 of the HSV-TK gene can mediate proper processing andtransport of human β-globin-like transcripts in the absence of splicing.

6. HSV-TK PPE Functions in an Orientation-dependent Manner

To determine whether this novel sequence element functions in anorientation-dependent manner, we constructed the plasmidspTK119ASβ1(-)2(-) and pTK119ASβ1(+)2(+) in which HSV-TK nt 361-to-479were inserted at the NcoI site of the human β-globin gene, but in theanti-sense orientation (FIG. 3). FIG. 3 is a summary of the structuresof plasmids containing an insertion of the HSV-TK element in the senseversus anti-sense orientation or in the 5'- versus 3'-UTR of theβ-globin gene and the data obtained with these plasmids. The arrowsindicate the orientation of the HSV-TK sequences. All other symbols arethe same as those described in FIG. 1. The data summarized on the rightwere obtained as described in FIG. 1.

The data reported in FIG. 3 demonstrated that cells transfected withpTK119β1(-)2(-) efficiently accumulated the chimeric RNA, while cellstransfected with pTK119ASβ1(-)2(-) failed to do so. Thus, theorientation of this inserted HSV-TK sequence element is important,consistent with it functioning at the RNA level.

7. Effect of Duplication of this HSV-TK PPE

Although insertion of HSV-TK nt 361-to-479 into the NcoI site of thehuman β-globin gene leads to fairly efficient rescue of the defects inprocessing of intronless β-globin-like transcripts, it does not enablecytoplasmic accumulation of globin RNA to the levels obtained when anearly full-length copy of the transcribed region of the HSV-TK gene isinserted into the β-globin gene (FIGS. 1 and 2). One hypothesis toexplain this finding is that more than one PPE is needed for maximallyefficient RNA processing. To test this hypothesis, we constructedplasmids p2xTK119β1(-)2(-) and p2xTK119β1(+)2(+) that contain two tandemcopies of this 119-bp HSV-TK sequence inserted at the NcoI site of thehuman β-globin gene. Duplication of this HSV-TK PPE resulted in at leasta 2-fold increase in cytoplasmic accumulation of globin-like RNA in theabsence of introns (FIG. 4). This level is comparable to the levelobtained by insertion of the entire coding region of the HSV-TK gene(FIG. 4 vs. FIG. 1A).

FIG. 4 is a summary of the structures of the plasmids containing tandemcopies of HSV-TK nt 361-to-479 and the data obtained with theseplasmids. BH indicates BspHI. All other symbols are the same as thosedescribed in the legend to FIG. 1. The data summarized on the right wereobtained as in FIG. 1.

Greenspan and Weissman (1985, supra) and Buchman and Berg (1988, supra)had noted previously that non-overlapping sequences transcribed from theHSV-TK gene can accumulate in vivo in the absence of intron excision. Wenow reinterpret this finding to indicate that the HSV-TK transcribedregion contains at least two PPEs, rather than no negative, cis-actingelement requiring an intron for proper processing and transport. Ourfinding also demonstrates that two copies of the PPE we have mapped herecan function at least additively in permitting intron-independent geneexpression.

8. Mutations in this PPE that Affect its Ability to MediateIntron-independent Gene Expression

To identify bases within this 119-bp HSV-TK sequence element requiredfor intron-independent gene expression, we constructed the plasmidspTK119LS0 β1(-)2(-), pTK119LS1β1(-)2(-), and pTK119LS2β1(-)2(-) andtheir corresponding intron-containing versions. These plasmids arederivatives of pTK119β1(-)2(-) and pTK119β1(+)2(+), respectively, intowhich linker-scanning substitution mutations had been introduced intothe nt 399-to-432 region of the inserted HSV-TK sequence (FIG. 5; Table1). Note that the nucleotide sequences of the entire 119 bp insert frompTK119LS0, pTK119LS1, and pTK119LS2 are listed as SEQ ID NOs:10, 11 and12, respectively.

FIG. 5 is a summary of the structures of the plasmids containinglinker-scanning mutations in the inserted 119-bp HSV-TK sequence elementand the abilities of these variants to enable intron-independent geneexpression. The schematic diagrams on the left indicate the sequence ofthe NruI-to-XhoI region of the 119-bp HSV-TK sequence element present ineach plasmid; the underlined, bold-faced letters highlight the alterednucleotides. All other symbols are the same as those described inFIG. 1. The data summarized on the right were obtained as in FIG. 1.

These linker-scanning mutants were found to mediate intron-independentgene expression. Whereas the mutations introduced in pTK119LS0 β1(-)2(-)led to an order-of-magnitude reduction in the cytoplasmic accumulationof the chimeric RNA, those introduced in pTK119LS1β1(-)2(-) andpTK119LS2β1(-)2(-) resulted in a reduction of only 2- to 3-fold. Thus,we conclude that specific bases within nt 399-to-432 of this 119-bpHSV-TK sequence are important for intron-independent expression of thehuman β-globin gene.

9. A Cellular 68 kDa Protein Specifically Binds to RNA Containing this119-nt HSV-TK Sequence

Pre-mRNAs are usually associated with distinct sets of heterogeneousnuclear ribonucleoproteins (hnRNPs) in the nucleus. To look for nucleartrans-acting factors that bind specifically to RNA corresponding totranscripts of the 119-bp HSV-TK sequence element (i.e., rTK119), wesynthesized radiolabeled rTK119 using T7 polymerase and the plasmidpT7/TK119. The labeled transcripts were purified, incubated with extractmade from nuclei of HeLa or COS cells, and exposed to UV light tocrosslink the bound protein to the radiolabeled RNA. After digestion ofthe unprotected RNA with RNase A, the RNA-protein adducts were resolvedby SDS-PAGE.

Several proteins were found to crosslink with rTK119. However, only thebinding of the approximately 68 kDa one was competed in asequence-specific manner (data not shown). The intensity of the otherabundantly crosslinked factor, corresponding to a protein approximately34 kDa in size, varied between experiments and cell extracts. Quitelikely, this latter band corresponds to a proteolytic product of the 68kDa protein or a non-specific crosslinking product.

10. Binding of this 68 kDa Protein to rTK119 Correlates with the Abilityof this PPE to Enable Cytoplasmic Accumulation of mRNA in vivo

UV-crosslinking analysis performed with deleted variants of rTK119indicated that the region of this RNA critical for efficient in vitrobinding of the 68 kDa protein is located around nt 387-to-419 (Liu,"Effects of intron and exon sequence elements on intron-dependent andintron-independent gene expression," Ph.D. Thesis. University ofWisconsin-Madison, Madison, Wis., 1994). This same region is alsoessential for PPE function in vivo (summarized in FIG. 2). To furtherassess the biological importance of the binding of the 68 kDa protein torTK119, we examined whether binding of this protein to RNAs containingalterations in this sequence (FIG. 6) correlated with the previouslydetermined abilities of the sequence to enable intron-independent geneexpression (FIG. 5).

FIG. 6 is a set of schematic diagrams of the transcribed regions of theplasmids with linker-scanning mutations in the PPE used to synthesizethe radiolabeled rTK119, rTK119LSO, rTK119LS1, and rTK119LS2 RNAs and asummary of the UV-crosslinking data obtained with these RNAs. The firstcolumn on the right indicates the binding of the 68 kDa protein to eachof these RNAs relative to its binding to rTK119 as determined byquantitative analysis with a PhosphorImager of two independentexperiments similar to the one shown in panel B. The last column istaken from the data in FIG. 5.

To assay for the abilities of these mutant RNAs to bind the 68 kDaprotein, we first constructed the plasmids pT7/TK119LSO, pT7/TK119LS1,and pT7/TK119LS2 (FIG. 6). These plasmids are identical in sequence topT7/TK119 except for the replacement of the NruI-to-XhoI region with thecorresponding region from plasmids pTK119LS0β1(-)2(-),pTK119LS1β1(-)2(-), and pTK119LS2β1(-)2(-) (FIG. 5), respectively. Theseplasmid DNAs were cleaved with NcoI and transcribed with T7 RNApolymerase in parallel reactions to make rTK119, rTK119LS0, rTK119LS1,and rTK119LS2 radiolabeled to similar specific activities. Identicalamounts of each RNA were incubated with equal amounts of HeLa cellnuclear extract and processed as in the UV-crosslinking experimentsdescribed above. The relative abilities of these RNAs to bind the 68 kDaprotein were determined from the relative intensities of the RNA-proteinadducts (data not shown). Quantitative analysis of these data indicatedthat the efficiencies with which these mutant RNAs bind the 68 kDaprotein (FIG. 6) correlate well with their abilities to enablecytoplasmic accumulation of intronless globin-like RNA (FIG. 5).

If sequence-specific binding of the 68 kDa protein to rTK119 isresponsible for enabling proper processing and cytoplasmic accumulationof intronless TK119-globin chimeric transcripts, this protein would beexpected to bind transcripts synthesized from pTK119β1(-)2(-), but notones synthesized from pβ1(-)2(-). To test this hypothesis, radiolabeledRNAs corresponding to these transcripts were synthesized with T7 RNApolymerase, incubated with HeLa cell nuclear extract in the presence ofthe non-specific competitor RNA, pβ19, and exposed to UV-light asdescribed above. Whereas transcripts containing this 119-nt HSV-TKsequence specifically crosslinked with the 68 kDa protein in vitro, theintronless β-globin transcripts did not (data not shown). These dataindicate that the pre-mRNAs synthesized from pβ1(-)2(-) andpTK119β1(-)2(-) are differentially bound by the 68 kDa protein, with thepresence of the 119-nt HSV-TK sequence being responsible for thisdifference. Thus, we conclude that binding of this cellular 68 kDaprotein to this PPE probably plays an important role in the properprocessing and transport of these intronless transcripts. As we showelsewhere (Liu and Mertz, 1995, supra) this 68 kDa protein is hnRNP L,an abundant, cellular protein (Pinol-Roma, et al., Genes and Devel.2:215-227, 1988).

11. The Intronless Cellular Gene c-jun can Also Enable EfficientAccumulation of Intronless β-globin-like RNA

Although viral in origin, the HSV-TK PPE identified here is recognizedin a sequence-specific manner by hnRNP L, an abundant, 68 kDa cellularprotein, and can efficiently perform its function in mammalian cells inthe absence of any virally encoded proteins (Liu and Mertz, 1995,supra). Thus, it is likely that at least some cellular genes alsocontain similar sequence elements. The cellular gene c-jun is naturallyintronless, yet efficiently expressed in higher eukaryotes. To testwhether this gene might also contain elements functionally similar tothe HSV-TK PPE identified here, we inserted part of its coding region(nt 975-to-1963 relative to the transcription initiation site) into the5'-untranslated region of cDNA and genomic versions of the humanβ-globin gene.

FIG. 7 is a diagram of structures of the plasmids containing aninsertion of part of the coding region of the c-jun gene into the5'-untranslated region of the human β-globin gene and a summary of thedata obtained with them. Only the transcribed region of each gene isshown. Stippled rectangles indicate sequences from the transcribedregion of the c-jun gene; numbers at ends of stippled rectanglesindicate endpoints of the c-jun sequence inserted in nt relative to thec-jun gene's transcription initiation site. All other symbols are asdescribed in the legend to FIG. 1.

Analysis of the jun-globin hybrid RNA accumulated in CV-1PD cellstransfected with these chimeric genes indicated that sequence from c-juncan also enable intron-independent processing and/or transport ofβ-globin-like RNA (summarized in FIG. 7). Therefore, the transcribedregion of the cellular gene c-jun probably also contains at least onesequence element functionally similar to the HSV-TK PPE.

a. Role of HSV-TK PPE in Intron-independent Gene Expression

Much evidence indicates that the requirement of introns for theefficient expression of intron-dependent genes is post-transcriptionalin nature (Gruss, et al., 1979, supra; Hamer and Leder, 1979, supra;Buchman and Berg, 1988, supra; Ryu and Mertz, 1989, supra; Collis, etal., 1990, supra; Huang and Gorman, 1990, supra; Nesic, et al., 1993,supra). The HSV-TK PPE identified here probably also actspost-transcriptionally. First, the presence of this element in anintron-containing gene has little effect on cytoplasmic accumulation ofthe resulting mRNA. Second, the functioning of this element isorientation dependent. Third, transcripts containing this sequencespecifically interact with a 68 kDa protein present in nuclear extracts(FIG. 6). Fourth, a good correlation exists between binding of this 68kDa protein, hnRNP L, to this RNA element and its ability to function invivo (FIG. 6) (Liu and Mertz, 1995, supra). Thus, although we have notyet definitively eliminated the possibility that this element acts intranscription, a post-transcriptional mechanism is much more likely.

Transcripts synthesized from intronless variants of intron-requiringgenes are retained in the nucleus where they are degraded (FIG. 1; Ryuand Mertz, 1989, supra; Collis, et al., 1990, supra; Huang and Gorman,1990, supra, and references cited therein). One hypothesis to explainnuclear retention is that specific sequence elements present intranscripts prevent nucleocytoplasmic transport by binding proteinsrestricted to the nucleus. One such cis-acting sequence element otherthan splicing signals that is responsible for nuclear retention ofpre-mRNAs has been identified in HIV; Brighty and Rosenberg, Proc. Natl.Acad. Sci. USA 91:8314-8318, 1994). Nuclear retention can then beovercome by the interactions of trans-acting factors that enable nuclearexport (e.g., the HIV-encoded protein rev) with specific cis-actingsequences present in these transcripts (e.g., RRE). Therefore, specificRNA sequence elements exist that can either positively or negativelyregulate nuclear export of mRNAs.

In the presence of the HSV-TK PPE, intronless globin-like transcriptsare both stabilized in the nucleus and exported to the cytoplasm (FIG.1B; Ryu, 1989, supra). However, stabilization of RNA in the nucleus neednot imply nucleocytoplasmic transport. For example, unspliced HIVtranscripts are stabilized, yet restricted to the nuclei of COS cells inthe absence of rev (Cullen, et al., 1988, supra; Emerman, et al., 1989,supra; Felber, et al., 1989, supra; Hammarskjold, et al., 1989, supra;Malim, et al., 1989, supra). In human T cells, these same transcriptsare degraded (Malim and Cullen, Mol. Cell. Biol. 13:6180-6189, 1993).The presence of rev not only enables the nucleocytoplasmic transport ofunspliced viral mRNAs, but also acts to stabilize these mRNAs in T cellnuclei.

The mechanism of nuclear stabilization remains unclear. Lu, et al.(1990, supra) have shown that the presence of the tat/rev 5' splice siteis essential for the nuclear stability of unspliced transcripts of HIV.We have noted that nts 435-443 (AGG/GUGAGA) of the HSV-TK PPE sharesignificant homology to the 5' splice site consensus sequence. However,(i) a nt 438 (GT-AT) point mutation, predicted to inactive this putativesplice site-like sequence, does not affect its function, and (ii)transcripts synthesized from pTK69 β1(-)2(-) contain this sequence, yetfail to accumulate in the cytoplasm. Therefore, this HSV-TK PPEfunctions independently of this putative 5' splice sequence. Thus, weconclude that interaction of this HSV-TK PPE sequence with hnRNP L isprobably responsible for nuclear stabilization of the intronlessTK-globin transcripts.

One plausible hypothesis is that stabilization and nuclear export areconsequences of proper 3'-end formation. The presence of introns hasbeen shown to stimulate cleavage and polyadenylation of many pre-mRNAsin vitro (Niwa, et al., 1990, supra; Niwa and Berget, Genes & Dev.5:2086-2095, 1991) and in vivo (Collis, et al., 1990, supra; Pandey, etal., 1990, supra; Chiou, et al., J. Virol 65:6677-85, 1991; Nesic, etal., 1993, supra; Liu, 1994, supra; Nesic and Maquat, Genes & Dev.8:363-375, 1994). Transport of intronless histone transcripts isstimulated by proper 3'-end formation (Eckner, et al., EMBO. J.10:3513-3522, 1991). We find that the presence of this HSV-TK PPE inintronless β-globin transcripts leads to an increase in the accumulationof polyadenylated β-globin-like RNA (unpublished observation). Thus,this HSV-TK PPE might function primarily to facilitate 3'-end formation.However, our finding that it functions well in the 5'-UTR, but not wheninserted close (e.g., 21 bases) to the polyadenylation signal (data notshown), does not support this simple model.

b. RNA-protein Interactions in Pre-mRNA Processing

Nascent transcripts rapidly associate with hnRNP proteins and snRNPs ina sequence-specific manner (Bennett, et al., Mol. Cell. Biol.12:3165-3175, 1992; Dreyfuss, et al., Rev. Biochem. 62:289-321, 1993;Matunis, et al., J. Cell. Biol. 121:219-228, 1993). The specificarrangement of hnRNPs on a transcript is likely an important determinantof the subsequent steps in mRNA biogenesis and transport. For example,Swanson and Dreyfuss (1988, supra) have shown that the major hnRNPproteins A1, C, and D specifically bind to the 3'-ends of the introns ofhuman β-globin pre-mRNA; however, no specific, high-affinity bindingsites for these proteins exist on intronless globin RNAs. Using aUV-crosslinking assay, we observed only one significant difference inthe pattern of proteins bound to the intronless β-globin transcriptscontaining the 119-nt HSV-TK sequence versus ones lacking it, i.e., thepresence of a band corresponding to a protein 68 kDa in size (data notshown). Immunoprecipitation with an anti-hnRNP L-specific serum anddirect UV-crosslinking experiments with recombinant hnRNP L indicatedthat the 68 kDa protein is hnRNP L (data not shown). Binding of hnRNP Lto the transcript was found to correlate with efficient processing andtransport of the RNA (data not shown). Thus, we conclude that thebinding of hnRNP L probably plays an important role in the properprocessing and transport of intronless human β-globin mRNA containingthis HSV-TK PPE.

Several hnRNP proteins have been shown to bind RNA in asequence-specific manner. For example, hnRNP A1 binds to 5' splicesite-like sequences (Burd and Dreyfuss, EMBO. J. 13:1197-204, 1994),hnRNP I binds to polypyrimidine tract sequences (Garcia-Blanco, et al.,Genes & Dev 3:1874-1886, 1989; Patton, et al., Genes & Dev. 5:1237-51,1991; Bennett, et al., 1992, supra; Ghetti, et al., Nucleic Acids Res.20:3671-8, 1992), and hnRNP K has a high affinity for poly(C)-richsequences (Swanson and Dreyfuss, Mol. Cell Biol. 8:2237-2241, 1988;Matunis, et al., 1992, supra). Specific RNA-protein interactions oftenregulate important steps in pre-mRNA processing. By mutational analysis,we showed here that hnRNP L binds to RNA with high sequence specificity.HnRNP L is an abundant, nuclear protein found in association with someof the nascent transcripts observed in the giant loops of lampbrushchromosomes of amphibian oocytes (Pinol-Roma, et al., J. Cell. Biol.109:2575-2587, 1989). At least some of the hnRNP L present in cellsexists free from association with previously defined hnRNP complexes(Pinol-Roma, et al., 1989, supra). The precise function of hnRNP L isnot yet known. One possibility is that hnRNP L functions to shuttlebetween the nucleus and cytoplasm RNAs to which it is bound, much likehnRNP A1 has been shown to do (Pinol-Roma and Dreyfuss, Nature355:730-732, 1992). A second possibility is that binding of this proteinin the 5'-UTR helps to insure the rapid formation of ribonucleoproteincomplexes on the nascent transcripts, thereby protecting the RNAs fromdegradation in the nucleus and, consequently, allowing the RNAs to beefficiently processed and, eventually, exported to the cytoplasm. Athird, and not mutually exclusive, possibility is that binding of hnRNPL may facilitate recruitment to the RNA of other hnRNP proteins that,subsequently, function in other steps in pre-mRNA processing (e.g.,polyadenylation) and transport to the cytoplasm.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAATCGCGAACATCTACACCACACA60                ACACCGCCTCGACCAGGGTGAGATATCGGCCGGGGACGCGGCGGTGGTAATGACAAGCG119                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TTAGGCAGAATCCAGATGCTCAAGGCC27                                                 (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ACATTTGCTTCTGACACAACTGTG24                                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATCTACACCACA12                                                                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TAGTAGATCTAGA13                                                               (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TCGCGAACATCTACACCACACAACACCGCCTCGA34                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCGCGAACTAGTAGATCTAGAAACACCGCCTCGA34                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TCGCGAACATCTACACCAGATCTCACCGCCTCGA34                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCGCGAACATCTACACCACACAAAGATCTCTCGA34                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAATCGCGAACTAGTAGATCTAGAA60                ACACCGCCTCGAGCAGGATGAGATATCGGCCGGGGACGCGGCGGTGGTAATGACAAGCG119                (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAATCGCGAACATCTACACCAGATC60                TCACCGCCTCGAGCAGGATGAGATATCGGCCGGGGACGCGGCGGTGGTAATGACAAGCG119                (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAATCGCGAACATCTACACCACACA60                AAGATCTCTCGAGCAGGATGAGATATCGGCCGGGGACGCGGCGGTGGTAATGACAAGCG119                __________________________________________________________________________

We claim:
 1. A chimeric RNA molecule comprising a 5' untranslatedregion, a 3' untranslated region and a protein-coding sequence, whereinthe 5' untranslated region or the 3' untranslated region of the RNAmolecule comprises at least one pre-mRNA processing element not nativelyconnected to the protein-coding sequence, wherein the pre-mRNAprocessing enhancer has a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, NO:11, and NO:12.
 2. A gene constructcomprising a DNA sequence encoding at least one pre-mRNA processingenhancer downstream from a promoter and upstream from a protein codingsequence, wherein the promoter is operably connected to theprotein-coding sequence, wherein the pre-mRNA processing enhancer is notnatively connected to the protein coding sequence and wherein thepre-mRNA processing enhancer increases the accumulation of the proteincoding sequence at least two-fold without requiring intron excision andwherein a single pre-mRNA processing enhancer is localized to anucleotide length of less than 300 nucleotides.
 3. A chimeric RNAmolecule comprising a 5' untranslated region, a 3' untranslated regionand a protein-coding sequence, wherein the 5' untranslated region of theRNA molecule comprises at least one pre-mRNA processing element notnatively connected to the protein-coding sequence and wherein thepre-mRNA processing enhancer is within the 5' untranslated region,additionally comprising a translation enhancer sequence upstream fromthe protein-coding sequence and downstream from the pre-mRNA processingenhancer.
 4. A gene construct comprising a DNA sequence encoding atleast one pre-mRNA processing enhancer, wherein said construct comprisesa unique restriction endonuclease site downstream from said DNA sequenceand wherein the restriction endonuclease site is part of a polylinker.